New vaccination approach using formalin-killed Streptococcus pyogenes vaccine on the liver of Oreochromis niloticus fingerlings

Newly synthesized vaccines prepared from formalin-killed bacteria Streptococcus pyogenes were investigated in the current study to evaluate the effectiveness of the newly synthesized vaccine as well as their safety by injected intraperitoneal. The study involved several steps 1st step is the preparation of the vaccine followed by the 2nd step: Evaluate the effectiveness and vaccine safety against pathogenic S. pyogenes through 4 different groups including control (Group I). Group II (Bacterial, infected group), Group III (Vaccine), and the Last group was the challenged group after the vaccination (Vacc + Bac). Different Immunological and biochemical parameters were measured in addition to hematological and histopathological examinations. For example, oxidative/antioxidants, inflammatory biomarkers, fragmentation and cell damage, and finally the histopathological study. The current study showed an increase in all oxidative, inflammatory, and cell damage (DNA fragmentation assays), additionally markedly elevation in histopathological cell damage in the infected group (Group II) compared with the control group. The vaccine and challenged after vaccination group (vaccine + Bacteria), showed great improvement in oxidative biomarkers (LPO) and an increase in antioxidants biomarkers (GSH, SOD, GST, DPPH, ABTS, GR and GPx), Also the inflammation and histopathological examination. The newly synthesized vaccine improved the resistance of Oreochromis niloticus and can be used as a preventive therapy agent for pathogenic bacteria S. pyogenes.

www.nature.com/scientificreports/such as endophthalmitis, exophthalmia, and eye hemorrhages.External bleeding can be seen most commonly around the mouth or at the base of the fins, as well as reddish pigmentation around the anus or on the genital papilla.Acute Streptococcus outbreaks are often accompanied by abdominal fluid.Ascites and anus protrusion are commonly seen together.One of the best methods for preventing and controlling viral diseases in fish is vaccination, which works by boosting the immune system 8 .Vaccines have been widely utilized in aquaculture as a defensive mechanism against various infections to prevent the host from infection 9 .In vaccines; pathogenic organisms are rendered non-pathogenic by various methods to produce antigens.Vaccines work by stimulating the immune system of animals to be more resistant to diseases caused by common pathogens in the future.Vaccine-stimulated antibody-producing cells (B lymphocytes) remain sensitized and ready to respond to the agent if it ever gains entry 10 .Vaccines are often manufactured by killing the infectious agent in formalin and using it as an antigen to stimulate the immune system.Aquaculture vaccines are mostly killed vaccines.In addition to being less expensive, easier to make, stable in storage, and free of virulence.Most of the time, these vaccines target the outer layer of microorganisms or inner sections without losing the ability to replicate in the host 11 .As a result, this study evaluated an inactivated S. pyogenes vaccine for Nile tilapia to control streptococcal disease outbreaks for the first time, which were indicated by hematological parameters, immune biomarkers, and DNA fragmentation assays.Furthermore, antioxidant enzymes can be used as immune enhancement detectors.

The study outcome
Our study aims to investigate an alternative therapy against microbial infections (S. pyogenes), by increasing Nile tilapia fish's natural immunity and reducing the pathogenicity of microbial infection using the formalin-killed bacteria vaccine technique.

Fish and rearing management
Healthy, 80 pathogen-free Nile tilapia fingerlings (15 ± 5) obtained from the National Institute of Oceanography and Fisheries (NIOF), were used in the current study.Nile tilapias were reared in 1000 L quarantine tanks in aerated fresh water at Marine Biotechnology and Natural Products Laboratory Animal House.A commercial diet (30% protein) was fed to the fish, which accounted for 4% of their total weight.Daily water cleaning and monitoring were conducted to maintain optimal fish conditions during the experiment, pH (7.5-8), ammonia (0.0016 ppm), dissolved oxygen (7 mg/l), and water temperature (28.1 C) were adjusted.The 25% of water beside the aeration was exchanged daily.Fish are housed for 4 weeks before the experiment begins to monitor their health.Three fish are necropsied randomly from each treatment.To ensure liver samples are free of S. pyogenes bacteria or other pathogens, samples are collected for bacteriological testing.The study is reported in accordance with ARRIVE guidelines and was approved by the animal ethics committee of the research ethics committee (ERC) of the Molecular Biology Research and Studies (MBRSI), Assiut University, Assiut, Egypt.With Rec.Number IOR G0010947-SCI-21-34-A".

Bacterial growth
One Litre of deionized water was combined with 15 g of pancreatic digest of casein, 5 g of peptic digest of soybean meal, 5 g of sodium chloride, and 15 g of agar to generate the growth medium.The components were combined by stirring the flask using a magnetic stirrer.The aliquot was divided into ten 100 ml flasks and autoclaved for 15 min at 121 °C in each of them to sterilize them.The flasks were preserved by being kept in a freezer at 4 °C.Trypticase Soy Agar (TSA) can be made into blood agar media by adding 5% sheep blood.Finally, S. pyogenes from the American Type Culture Collection (ATCC) In Virginia, United States, was added to the media.And media culture study was done.

Vaccine preparation
Streptococcus pyogenes strain used for this study was purchased with ATCC19615 according to the methods described by Klesius et al. 12 , Pretto-Giordano et al. 13 , and Bastardo et al. 14 .The Strain was cultured and fermented in tryptic soy broth at 30 C for 72 h using a freshly made culture from an ATCC sample.Ten percent formalin solution was added to the culture in TSB to inactivate it, and after 24 h at 4 °C, the final concentration was 3%.To insure that the bacterial cells are inactive form 0.1 ml of the formalin-treated cultured was streaked on sheep blood agar.After being centrifuged at 7000 g for 30 min.at 10 °C with the inactivated culture, the cell pellet was re-suspended in sterile normal saline.The cell pellet was once again re-suspended in TSB after a second round of centrifugation, resulting in a final concentration of 2.0 × 10 7 colony forming units (CFU) per milliliter.This CFU mL was calculated from colony counts following culture on TSA and measured using a spectrophotometer at a wavelength of 540 nm.

Vaccine safety assessment
Ten fish per group received 0.2 ml of adjuvant vaccine intraperitoneally (I.P) to examine the vaccines' potential side effects in vivo.MS 222 (50 ppm) was used to anesthetize the fish before usage 15 .Throughout the first week following injection, we notice any harmful effects, such as behavioral abnormalities, lesions close to the injection site, and immediate mortality.Necropsies were done on fish four and 8 weeks after they had been shot to assess the long-term consequences 16 .

Routine biochemical parameters
The total protein was determined according to the method described by Hiller et al. 17 , and the total lipid was determined according to the method described by Zollner and Krisch's 18 method.Additionally, the total glucose was determined according to the method of Richardson 19 , the total protein, total lipid and total glucose were determined according to previous methods using Bio-diagnostic kits.

Non-specific immune parameters
Nitric oxide (NO) NO was determined using the Greiss reagent and served as an indicator of No production according to Wang et al. 20 .

Oxidative/antioxidant biomarkers
The oxidative stress lipid peroxidation (LPO) was determined by measuring malondialdehyde (MDA) levels based on the reaction of MDA with Thiobarbituric acid according to Kei 22 .Different antioxidant biomarkers were evaluated in the current study Catalase (CAT): the assay was performed according to Hadwan, 23 .Superoxide dismutase (SOD) was determined by the indirect method using Pyrogallol as described by Marklund & Marklund 24 .Total reduced glutathione (GSH) the assay was performed according to Ellman 25 .Glutathione -S-transferase (GST) was determined according to the methods modified by Habig et al. 26 .Total glutathione reductase (GR) activity was determined by the method of Carlberg and Mannervik 27 .Assay of Glutathione Peroxidase (GPX) Glutathione peroxidase was determined by the method of JT 28 .

Diphenylamine assay of DNA fragmentation and Vitamin C
The Diphenylamine assays were described by Dische 29 and modified by Burton 30 .The level of Vitamin C was determined by the method of Omaye et al. 31 .

Histopathological examination
Liver tissue specimens were taken from all individual groups at the end of the experiential period according to the method described by Carleton et al. 32 .

Statistical analysis
For statistical analysis and graphing, Graph Pad PRISM software was used.A one-way analysis of variance was performed along with a Tukey post hoc test to determine the significance of the results.Alpha = 0.05 and denoted as P < 0.05 (*), P < 0.01 (**), or P < 0.001(***).

Ethical approval
The authors declare that they have conducted all applicable laws, guidelines, and regulations from the government.

Consent to participate
Authors informed.

Routine biochemical parameters
The Total protein levels were evaluated and showed a nonsignificant decrease in the bacterial group compared with the control group (0.08083 ± 0.01048 Vs 0.08983 ± 0.009827, P = 0.6650), additionally, the Vacc and Vacc + Bac showed significantly increased compared with control (0.2087 ± 0.03509, P = 0.0006***& 0.2482 ± 0.03260, P = 0.0006***).Furthermore, the Vacc and Vacc + Bac showed significant increases compared with the bacterial

Histopathology
Figure 6 shows the different histopathological changes in the liver of different experimental groups of Nile tilapia fingerlings.In the control group, there were no liver lesions and normal hepatocytes (A) were ordered and clustered in cords, separated by blood sinusoids.Additionally, polygonal hepatic cells and blood capillaries are seen to be organized normally.In the infected liver by S. pyogenes bacteria, severe damage, and inflammation (B) characterized by a red lump of blood (C) besides focal hemorrhages and inflammation are seen in dermal ulceration lesions.Free radicles may cause massive hemocyte aggregation (D) in the hepatic tissues because of stress and liver necrosis.The adjuvant and adjuvant + bacteria groups showing the arrangement of hepatocytes in the liver revealed vacuolation (E) and severe necrosis in some cells.Besides, lymphocytic aggregation's focal area (F) migrates from the dilated blood vessels toward the necrotic hepatic cells.

Discussion
The route of vaccine administration affects the quality and effectiveness of the vaccine, which is related to suitable immunization routes for stimulating the fish immune system, the intraperitoneal (IP) injection is considered the most effective route although it's stressful to the animals and hence expensive 33 .The continuous use of different antibiotics against different types of bacterial infections raises the phenomena of bacterial resistance resulting in super bacteria 34 .One of the possible solutions for such problems is the use of a vaccine as an alternative approach to treating aquaculture fish from pathogens although the effectiveness of vaccine there are different challenges such as specificity, pathogen, inoculation, and the lack of availability of different pathogens 35 .Pathogenic bacteria cause huge losses in the global aquaculture branch Streptococcus is one of the most serious bacterial infections at the economic level 36 .There are different Streptococcosis pathogenic in fish such as Streptococcus iniae, Streptococcus faecium, Streptococcus agalactiae, Streptococcus equi, Streptococcus equisimilis, and S. pyogenes 37 .Few studies investigated the destructive role of Streptococcus concerning vaccine and antagonistic activity and in vivo efficacy of natural immunity against streptococcal agents.And importantly to know that there is no previous study on S. pyrogen vaccine in fish 36 .
The hematological parameters of fish and aquatic animals can be used to observe their health status and immune responses.The WBC count is necessary for both the nonspecific and specific immune systems of fish 33 .A wide range of white blood cells collaborate in the cellular response, including, monocytes, and granulocytes.The current study showed that the vaccinated fish (21 days) showed a higher number of leukocytes (WBCs) than unvaccinated fish, the first group of vaccinated fish (21 days) showed a higher number than the second group of vaccinated fish (32 days) and the third group of vaccinated fish.The WBC counts of vaccinated Tilapia were higher before and after the S. pyogenes challenge.A higher WBC count leads to more antibodies, resulting in body resistance to extraneous substances 38 .An increase in phagocytic cells is associated with an increase in WBC count in the current experiment.On the other hand, the immunized fish showed a better immune response against infection with S. pyogenes.In the first group, WBC, Granulocytes (GRAN %, GRAN #), and Lymphocytes (LYM % and LYM #) counts were higher in fish injected with IP, while the same counts were also higher in fish treated with IP. (Fig. 5B, D, F, G) shows that non-vaccinated fish had lower WBC, granulocyte, and lymphocyte counts.Increasing WBC was attributed to the migration of white blood cells from the spleen to the blood circulation 39 .Despite being unclear in their function, lymphocytes in fish participate in inflammatory processes.The defense mechanisms might recruit them to the focus of the lysate, which explains their high numbers in the blood circulation of infected Tilapia.That's also consistent with our results which show significant increases in bacterial (infected group), but at lower levels than vaccinated groups.We hypothesized that after vaccine immunization, innate and specific immunity were activated.Many pathogenic organisms induce general and nonspecific stress and oxidative stress.According to several authors, bacterial challenge leads to anemia and impaired oxygen transport.Hematological parameters are a major indicator of bacterial infection in fish 40 .In the current study, HCT, HGB, and RBC significantly decreased in the bacterial group (Fig. 5H-J).By observing hematocrit, hemoglobin rate, and erythrocyte count, oxygen concentration can be related to fish health.According to Usman et al. 41 , with reduced erythrocyte count and hemoglobin rate, the infection impaired oxygen-carrying capacity in Tilapia infected by S. iniae.And that also seen in our study as hemoglobin was significantly decreased in fish with S. pyogenes.Possibly, pathogenic bacteria break down RBCs at a faster rate, and/ or fewer RBCs are formed 42 .According to Hardi et al. 43 , hemoglobin content decreases due to swelling of RBCs and poor hemoglobin mobilization of the spleen.The significant reduction of hemoglobin after fish infection could be attributed to severe anemia.As a result of the destruction of intestinal cells that produce vitamin B12, the hemoglobin portion of red cells 44 , the maximum value of MCV was found in the 1st (21 days) vaccinated group.The results also showed that the injection with the vaccine with varying times markedly increased MCV, while markedly decreasing the value of MCH and MCHC after infection of the Nile tilapia with S. pyogenes and injection of vaccine at 21 days was decreased while at 32-days vaccination (2nd group) MCH and MCHC was increased.WBC counts in the Nile tilapia fed different concentrations of vaccine were markedly higher, compared to the control group) According to Sutili et al. 45 , Platelets are classically known as essential mediators of hemostasis and thrombosis.Recently, platelets have been recognized for their inflammatory functions that modulate the immune response.The immunoreceptors on platelets allow them to recognize intravascular pathogens as sentinels.In response to activation, platelets release antimicrobial proteins (AMPs) and activate immune cells to clear pathogens.However, aberrant platelet activation can lead to inflammation and thrombotic events 46 .Platelet count increases under physiological conditions as a result of progenitor megakaryocyte reactivity, and during infection 47 .The RDW level increase has several clinical conditions, for example, in response to ineffective red cell ].uction, which can be caused by deficiencies in iron, vitamin B12, or folate.When compared with the bacterial (infected) group, the vaccinated group exhibited higher hematocrit levels and that can explain our results as the RDW was increased markedly in the group injected with the pathogen (GroupII) Fig. 5N and P. According to Santika et al. 48, the elevation of HCT levels of vaccinated Tilapia tends to induce an increase in HCT levels to a certain degree.A high HCT indicates healthy blood and the ability to bind oxygen.Both infection and injection vaccination treatments reduce hematocrit levels compared with controls (unvaccinated and uninfected).It is caused by the infection of S. pyogenes.According to Anderson et al. 49 , a decrease in HCT indicates a pathogen infection in fish.Additionally, the decreased hematocrit level after the bacterial infection is also due to decreased appetite after the S. pyogenes infection, low hematocrit can indicate a lack of protein, vitamin deficiencies, or infection of the fish.A marked elevation in the HCT value after the challenge test suggests that the blood cells play an important role in increasing the fast response in adequate quantities to relieve pathogenic bacteria infection.Tilapia had marked changes in HCT values on days 21 and 32 following vaccination.However, the RBCs were lower in fish vaccinated at 32 and elevated markedly after the challenge.Nevertheless, these values were similar to those observed in Tilapia with no stress stimulation in Martins et al. 50.The vaccinated fish had higher total RBCs and HGB percentages on day 21 than the bacterial group (injected with only bacteria) (Fig. 5J, H).Martins et al. 50identify Tilapia glycemia as a stress factor.Nevertheless, in this study, glucose was stable in all fish except those submitted to the challenge after vaccinated vaccination on 32 days (P = 0.008) Fig. 1A.Martins et al. 50, Okamura et al. 51 found similar indices among healthy Nile tilapia.There is evidence that high serum proteins are likely due to an improvement in fish non-specific immune responses 52,53 .
In the present study, the vaccinated groups (vaccinated only and challenged vaccinated after 32 days) markedly increased in reference to other groups (control and infected group) vaccinated group documented a notable (P = 0.0006***) elevation in total protein level, this finding in the present study agree Viji et al. 54 reported higher albumin level in biofilm groups than that of the control group Fig. 1C.The current studies evaluate the oxidative stress and antioxidant defense effects of vaccination against S. pyogenes in Nile Tilapia liver.The oxidative stress markers (Malondialdehyde), antioxidant defenses (SOD, CAT, GR, GSH, GPx, and GST) Fig. 2H, E, F,B, I and  G, and the total antioxidant capacity of Nile Tilapia were measured (DPPH and ABTS) Fig. 2C & D).Our study showed that Nile Tilapia vaccinated against S. pyogenes showed alterations in antioxidant defenses and oxidative stress.Additionally, the results revealed that vaccination against S. pyogenes caused liver tissue to undergo lipid peroxidation and that was confirmed and indicated in the histology study which showed several types of tissue damage and inflammatory response in reference to control (uninfected group) Fig. 4. It is important to note that, in vaccinated groups (with or without challenge), the immune system can reestablish its pro-and antioxidant balance after vaccination.Detection of antioxidant enzymes reveals the health of the body's antioxidant system, letting you know what the system is capable of when it comes to the digestion of oxygen-free radicals and protection of the tissue from oxidative damage.The rate of oxidative damage can be increased by many factors (disease, toxins, immunization, aging, and exercise), resulting in oxidative stress 55 .Depletion or elevation of ROS causes oxidative stress when the balance between oxidants and antioxidants is disrupted 56 .Free radicals are dangerous, but despite their potential danger, cells have various defense mechanisms against them 57 .The present study established that fish vaccinated against S. pyogenes have elevated levels of lipid peroxidation than fish immune systems 58 .SOD and CAT enzymes are the first line of defense against oxidative stress and are often used to measure ROS production 59 .The development of many vaccines to decrease the infection load as well as protect fish in aquaculture from acute bacterial infections such as Vibriosis, Streptococcus, Edwardsiellosis, Yersiniosis, and Furunculosis is ongoing 60 .After vaccination, there was a notable decrease in liver lipid peroxidation indicators observed in our study which showed a decrease in LPO in reference to the infected group with S. pyrogen Fig. 2A.The liver is a metabolically active and highly antioxidant organ.Consequently, compared to the other tissues, the liver of the infected group (the bacterial group) displayed greater levels of oxidative stress.The evidence from other studies agrees with our analysis 61 .The degree of oxidative damage under the influence of S. www.nature.com/scientificreports/pyogenes is demonstrated by the markedly higher levels of oxidative stress biomarkers in the liver of the infected group of bacteria than in the fish that received vaccinations.Skugor et al. 62 used multiple gene expression profiling to outline the mechanisms that determine the success of vaccine protection against pathogens like NF-kB.By upregulating NF-kB and AP-1 proteins in response to pathogens and cytokines, pathogens and cytokines induce a massive release of immune mediators and effector proteins into the body.Free radicals, and genotoxic agents, such as free radicals, are a few factors that damage cells, including NF-kB and Jun proteins, which affect cell growth, growth arrest, DNA repair, differentiation, and apoptosis.NF-kB can also activate protection against oxidative and cellular stress by providing anti-apoptotic and proliferation-promoting signals and that was in agreement with the current study which investigated the level of DNA damage in all groups where the bacterial group (infected) showed significant increases in DNA damage compared with control in contrast to vaccine group without challenge and that can be explained with the results of VitC in the current study which showed in Parallel to DNA damage results as shown in Fig. 3B, which indicted the DNA damage test showed a marked increase in the bacterial group while other group showed a non-markedly increased or decreased and that may be due to highly level of oxidative stress which lead to DNA damage as a defense mechanism the Vit C increase greatly to restore the GSH molecule which in turn restore the antioxidant enzymes to help to repair the cell damage and decreased oxidative stress 63,64 similar study for Skugor et al. 62 showed that genes for proteins involved in the regulation of redox status and protection against ROS had higher expression levels in vaccinated fish with high resistance to furunculosis 62 .ROS in cells may lead to an elevation of antioxidant enzymes as a defense mechanism.Providing cells with a comprehensive defense against ROS-induced damage is the primary function of antioxidants.There are several defense mechanisms included in these defenses, including low molecular weight compounds (e.g., glutathione and ascorbate), and our results indicate that GSH and Vitamin C levels are elevated in the vaccine group's Figs.2B and 3A 65 .Antioxidant enzymes (including SOD, GST, and GR) are also elevated.An important cofactor for several glutathione-dependent antioxidant enzymes is glutathione (GSH), a tripeptide that can neutralize ROS and act as a cofactor for glutathione (GSH).As observed in our study the GSH elevated greatly in vaccinated groups (without and with challenge) and decreased in the bacterial group (infected group) the same manner was observed in other antioxidant parameters such as GST, SOD, GR, and GPx 66 , (Fig. 2B, E-I).Antioxidant enzymes SOD, CAT, and GPx protect against oxidative stress by converting superoxide radicals into hydrogen peroxide, then water, and molecular oxygen.An important defense against oxidative stress in fish is the induction of antioxidant enzymes.Superoxide dismutase is a group of metalloenzymes responsible for dismutating superoxide into hydrogen peroxide in aerobic organisms and playing an essential antioxidant role 67 .Compared to the control group, the vaccinated group showed significantly higher SOD activity.Following immunization, an adaptive response could be observed that neutralizes ROS and also prevents membrane lipid peroxidation, especially when combining Fe 3+ and oxygen-generating systems.Tkachenko et al. 68 showed that It appears that, in response to oxidative stress, the decreased CAT activities in muscles and gills of vaccinated trout indicate that their capacity to scavenge hydrogen peroxide has been reduced.The three most important antioxidant enzymes that contribute to antioxidant defenses against oxidative stress are glutathione peroxidase, reductase, and transferase.As an antioxidant, GR plays an important role both in protecting cellular membranes from free radical damage as well as regulating metabolic pathways in a cell.In an NADPH-dependent reaction, glutathione disulfide is reduced to reduced glutathione when this enzyme catalyzes the reduction process.Pei et al. 69 reported that the liver had significantly higher GR activity than other tissues, most likely due to the availability of NADPH in the liver tissues 69 .A variety of enzymes are capable of inactivating lipid-derived hydroperoxides, including selenoperoxidases.There are two types of selenoperoxidases in cells: classical GSH-peroxidase (GPx), which acts on relatively polar substrates like H 2 O 2 and fatty acid hydroperoxides, and phospholipid hydroperoxide GSH-peroxidase.The glutathione disulfide-dependent GR is required for GPx.Oxidative stress and cytotoxicity result from a decrease in glutathione-mediated antioxidant defenses, whereas elevated levels of intracellular GSH are regarded as adaptive responses to oxidative stress.Comparing Tilapia vaccinated with the bacterial group, the total antioxidant capacity in the liver was significantly higher.As a result of impairment in enzymatic and non-enzymatic antioxidant synthesis, levels of cellular total antioxidant may be reduced in the infected group 70 .In vaccinated fish, glutathione-mediated antioxidant defense as well as endogenous CAT is critical for intracellular antioxidant defense.However, the antioxidant defenses of vaccinated fishes were markedly higher, probably due to liver functional activity.In Tilapia, glutathione-mediated antioxidant defenses were also demonstrated to protect against endosulfan-induced oxidative stress.Analysis of oxidative stress parameters showed marked differences among fish vaccinated against S. pyogenes.Oxidative stress and tissue responses are strongly correlated.Infected fish (bacterial group) (Fig. 2A showed an elevated level of lipid and protein oxidation biomarkers (Fig. 1B) 62 .A vaccination increased liver antioxidant defenses and made liver tissue more susceptible to oxidative damage Fig. 4 The results suggest that vaccination against S. pyogenes induced oxidative stress.A vaccination can restore liver tissue's pro-and antioxidant balance.Infection and imbalance of the immune system through infectious agents result in an elevation of inflammatory systemic response 68 , and that showed in the levels of MPO and NO results Fig. 6A and B, leading to an elevation of LPO.Consequently, inflammation induced by infectious stimuli involves the reciprocal control of major mediators (COX-2, NO, ROS, and glutathione).In agreement with our results, COX-2 converts arachidonic acid into inflammatory prostaglandins with the consequent release of cytokines in consequence leads to elevated levels of both NO and MPO which were elevated in the infected group (Fig. 6A, B), and decreased markedly in the vaccinated group.There are many mechanisms behind liver tissue injury.The main cause of liver injury is free radicals 71 .They damage biological membranes by lipid peroxidation, resulting in liver cell degeneration and necrosis, and an increase in lipid peroxides in the body.MDA is the product of lipid peroxidation.Deficiency in anti-oxidants is indirectly related to membrane peroxidation and dehydration.A continuous invasion of streptococci scavenges MDA by antioxidant enzyme systems, which are products of lipid peroxidation.Indirectly, its content reflects the body's antioxidant capacity and levels of membrane peroxidation.Antioxidant enzymes begin to play an increasingly important role in radical scavenging as Streptococci continue to invade.As infection time extended, antioxidants and other activities decreased, aggravating liver damage 72 .

Conclusion
The current study focuses on the effect of vaccine (formalin-killed bacteria) before and after infection on Nile tilapia immune enhancement and bacteria resistance.Considering the hematological parameters, immunological responses, and protection results in this study, we concluded that the vaccine has the effect of enhancing protective immunity against S. pyogenes.

Figure 1 .
Figure 1.Different biochemical parameters (A) glucose (B) Total lipid and (C) total protein, Where the Con.The control (without treatment), Bac (the infected group with S. pyogenes), Vacc (the vaccinated group),Vacc + Bacc (Challenged group after vaccination).

Figure 4 .
Figure 4. Showed Vit C (A) and DNA fragmentation (B) Where the Con.The control (without treatment), Bac (the infected group with S. pyogenes), Vacc (the vaccinated group), Vacc + Bacc (Challenged group after vaccination).

Figure 5 .
Figure 5.The different Hematological parameters indices in four experimental groups, Where the Con.The control (without treatment), Bac (the infected group with S. pyogenes), Vacc (the vaccinated group), Vacc + Bacc (Challenged group after vaccination).